METAPHOR: Metabolic evaluation through phasor-based hyperspectral imaging and organelle recognition for mouse blastocysts and oocytes.

Only 30% of embryos from in vitro fertilized oocytes successfully implant and develop to term, leading to repeated transfer cycles. To reduce time-to-pregnancy and stress for patients, there is a need for a diagnostic tool to better select embryos and oocytes based on their physiology. The current standard employs brightfield imaging, which provides limited physiological information. Here, we introduce METAPHOR: Metabolic Evaluation through Phasor-based Hyperspectral Imaging and Organelle Recognition. This non-invasive, label-free imaging method combines two-photon illumination and AI to deliver the metabolic profile of embryos and oocytes based on intrinsic autofluorescence signals. We used it to classify i) mouse blastocysts cultured under standard conditions or with depletion of selected metabolites (glucose, pyruvate, lactate); and ii) oocytes from young and old mouse females, or in vitro-aged oocytes. The imaging process was safe for blastocysts and oocytes. The METAPHOR classification of control vs. metabolites-depleted embryos reached an area under the ROC curve (AUC) of 93.7%, compared to 51% achieved for human grading using brightfield imaging. The binary classification of young vs. old/in vitro-aged oocytes and their blastulation prediction using METAPHOR reached an AUC of 96.2% and 82.2%, respectively. Finally, organelle recognition and segmentation based on the flavin adenine dinucleotide signal revealed that quantification of mitochondria size and distribution can be used as a biomarker to classify oocytes and embryos. The performance and safety of the method highlight the accuracy of noninvasive metabolic imaging as a complementary approach to evaluate oocytes and embryos based on their physiology.

Hyperspectral oblique plane microscopy enables spontaneous, label-free imaging of biological dynamic processes in live animals.

Spontaneous Raman imaging has emerged as powerful label-free technique for investigating the molecular composition of medicines and biological specimens. Although Raman imaging can facilitate understanding of complex biological phenomena in vivo, current imaging modalities are limited in speed and sample compatibility. Here, we introduce a single-objective line-scanning light-sheet microscope, named [Formula: see text]-OPM, which records Raman images on a timescale of minutes to seconds. To demonstrate its function, we use [Formula: see text]-OPM to map and identify microplastic particles based on their Raman spectral characteristics. In live zebrafish embryos, we show that [Formula: see text]-OPM can capture wound dynamics at five-minute intervals, revealing rapid changes in cellular and extracellular matrix composition in the wounded region. Finally, we use [Formula: see text]-OPM to synchronize and average 36,800 individual frames to obtain hyperspectral videos of a zebrafish embryo's beating heart at an effective 28 frames per second, recording compositional changes throughout the cardiac cycle.

Localization of unlabeled bepirovirsen antisense oligonucleotide in murine tissues using in situ hybridization and CARS imaging.

Current methods for detecting unlabeled antisense oligonucleotide (ASO) drugs rely on immunohistochemistry (IHC) and/or conjugated molecules, which lack sufficient sensitivity, specificity, and resolution to fully investigate their biodistribution. Our aim was to demonstrate the qualitative and quantitative distribution of unlabeled bepirovirsen, a clinical stage ASO, in livers and kidneys of dosed mice using novel staining and imaging technologies at subcellular resolution. ASOs were detected in formalin-fixed paraffin-embedded (FFPE) and frozen tissues using an automated chromogenic in situ hybridization (ISH) assay: miRNAscope. This was then combined with immunohistochemical detection of cell lineage markers. ASO distribution in hepatocytes versus nonparenchymal cell lineages was quantified using HALO AI image analysis. To complement this, hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) imaging microscopy was used to specifically detect the unique cellular Raman spectral signatures following ASO treatment. Bepirovirsen was localized primarily in nonparenchymal liver cells and proximal renal tubules. Codetection of ASO with distinct cell lineage markers of liver and kidney populations aided target cell identity facilitating quantification. Positive liver signal was quantified using HALO AI, with 12.9% of the ASO localized to the hepatocytes and 87.1% in nonparenchymal cells. HS-CARS imaging specifically detected ASO fingerprints based on the unique vibrational signatures following unlabeled ASO treatment in a totally nonperturbative manner at subcellular resolution. Together, these novel detection and imaging modalities represent a significant increase in our ability to detect unlabeled ASOs in tissues, demonstrating improved levels of specificity and resolution. These methods help us understand their underlying mechanisms of action and ultimately improve the therapeutic potential of these important drugs for treating globally significant human diseases.

Optical sectioning of unlabeled samples using bright-field microscopy.

The bright-field (BF) optical microscope is a traditional bioimaging tool that has been recently tested for depth discrimination during evaluation of specimen morphology; however, existing approaches require dedicated instrumentation or extensive computer modeling. We report a direct method for three-dimensional (3D) imaging in BF microscopy, applicable to label-free samples, where we use Köhler illumination in the coherent regime and conventional digital image processing filters to achieve optical sectioning. By visualizing fungal, animal tissue, and plant samples and comparing with light-sheet fluorescence microscopy imaging, we demonstrate the accuracy and applicability of the method, showing how the standard microscope is an effective 3D imaging device.

ChiSCAT: Unsupervised Learning of Recurrent Cellular Micromotion Patterns from a Chaotic Speckle Pattern.

There is considerable evidence that action potentials are accompanied by "intrinsic optical signals", such as a nanometer-scale motion of the cell membrane. Here we present ChiSCAT, a technically simple imaging scheme that detects such signals with interferometric sensitivity. ChiSCAT combines illumination by a chaotic speckle pattern and interferometric scattering microscopy (iSCAT) to sensitively detect motion in any direction. The technique features reflective high-NA illumination, common-path suppression of vibrations, and a large field of view. This approach maximizes sensitivity to motion, but does not produce a visually interpretable image. We show that unsupervised learning based on matched filtering and motif discovery can recover underlying motion patterns and detect action potentials. We demonstrate these claims in an experiment on blebbistatin-paralyzed cardiomyocytes. ChiSCAT opens the door to action potential measurement in scattering tissue, including a living brain.

Deep learning-based segmentation of subcellular organelles in high-resolution phase-contrast images.

Although quantitative analysis of biological images demands precise extraction of specific organelles or cells, it remains challenging in broad-field grayscale images, where traditional thresholding methods have been hampered due to complex image features. Nevertheless, rapidly growing artificial intelligence technology is overcoming obstacles. We previously reported the fine-tuned apodized phase-contrast microscopy system to capture high-resolution, label-free images of organelle dynamics in unstained living cells (Shimasaki, K. et al. (2024). Cell Struct. Funct., 49: 21-29). We here showed machine learning-based segmentation models for subcellular targeted objects in phase-contrast images using fluorescent markers as origins of ground truth masks. This method enables accurate segmentation of organelles in high-resolution phase-contrast images, providing a practical framework for studying cellular dynamics in unstained living cells.Key words: label-free imaging, organelle dynamics, apodized phase contrast, deep learning-based segmentation.

A high-resolution phase-contrast microscopy system for label-free imaging in living cells.

Cell biologists have long sought the ability to observe intracellular structures in living cells without labels. This study presents procedures to adjust a commercially available apodized phase-contrast (APC) microscopy system for better visualizing the dynamic behaviors of various subcellular organelles in living cells. By harnessing the versatility of this technique to capture sequential images, we could observe morphological changes in cellular geometry after virus infection in real time without probes or invasive staining. The tune-up APC microscopy system is a highly efficient platform for simultaneously observing the dynamic behaviors of diverse subcellular structures with exceptional resolution.

Dissecting lipid droplet biology with coherent Raman scattering microscopy.

Lipid droplets (LDs) are lipid-rich organelles universally found in most cells. They serve as a key energy reservoir, actively participate in signal transduction and dynamically communicate with other organelles. LD dysfunction has been associated with a variety of diseases. The content level, composition and mobility of LDs are crucial for their physiological and pathological functions, and these different parameters of LDs are subject to regulation by genetic factors and environmental inputs. Coherent Raman scattering (CRS) microscopy utilizes optical nonlinear processes to probe the intrinsic chemical bond vibration, offering label-free, quantitative imaging of lipids in vivo with high chemical specificity and spatiotemporal resolution. In this Review, we provide an overview over the principle of CRS microscopy and its application in tracking different parameters of LDs in live cells and organisms. We also discuss the use of CRS microscopy in genetic screens to discover lipid regulatory mechanisms and in understanding disease-related lipid pathology.

Viewing early life without labels: optical approaches for imaging the early embryo†.

Embryo quality is an important determinant of successful implantation and a resultant live birth. Current clinical approaches for evaluating embryo quality rely on subjective morphology assessments or an invasive biopsy for genetic testing. However, both approaches can be inherently inaccurate and crucially, fail to improve the live birth rate following the transfer of in vitro produced embryos. Optical imaging offers a potential non-invasive and accurate avenue for assessing embryo viability. Recent advances in various label-free optical imaging approaches have garnered increased interest in the field of reproductive biology due to their ability to rapidly capture images at high resolution, delivering both morphological and molecular information. This burgeoning field holds immense potential for further development, with profound implications for clinical translation. Here, our review aims to: (1) describe the principles of various imaging systems, distinguishing between approaches that capture morphological and molecular information, (2) highlight the recent application of these technologies in the field of reproductive biology, and (3) assess their respective merits and limitations concerning the capacity to evaluate embryo quality. Additionally, the review summarizes challenges in the translation of optical imaging systems into routine clinical practice, providing recommendations for their future development. Finally, we identify suitable imaging approaches for interrogating the mechanisms underpinning successful embryo development.

Investigation on lysosomal accumulation by a quantitative analysis of 2D phase-maps in digital holography microscopy.

Lysosomes are the terminal end of catabolic pathways in the cell, as well as signaling centers performing important functions such as the recycling of macromolecules, organelles, and nutrient adaptation. The importance of lysosomes in human health is supported by the fact that the deficiency of most lysosomal genes causes monogenic diseases called as a group Lysosomal Storage Diseases (LSDs). A common phenotypic hallmark of LSDs is the expansion of the lysosomal compartment that can be detected by using conventional imaging methods based on immunofluorescence protocols or overexpression of tagged lysosomal proteins. These methods require the alteration of the cellular architecture (i.e., due to fixation methods), can alter the behavior of cells (i.e., by the overexpression of proteins), and require sample preparation and the accurate selection of compatible fluorescent markers in relation to the type of analysis, therefore limiting the possibility of characterizing cellular status with simplicity. Therefore, a quantitative and label-free methodology, such as Quantitative Phase Imaging through Digital Holographic (QPI-DH), for the microscopic imaging of lysosomes in health and disease conditions may represent an important advance to study and effectively diagnose the presence of lysosomal storage in human disease. Here we proof the effectiveness of the QPI-DH method in accomplishing the detection of the lysosomal compartment using mouse embryonic fibroblasts (MEFs) derived from a Mucopolysaccharidosis type III-A (MSP-IIIA) mouse model, and comparing them with wild-type (WT) MEFs. We found that it is possible to identify label-free biomarkers able to supply a first pre-screening of the two populations, thus showing that QPI-DH can be a suitable candidate to surpass fluorescent drawbacks in the detection of lysosomes dysfunction. An appropriate numerical procedure was developed for detecting and evaluate such cellular substructures from in vitro cells cultures. Results reported in this study are encouraging about the further development of the proposed QPI-DH approach for such type of investigations about LSDs.

AutoMitoNetwork: Software for analyzing mitochondrial networks in autofluorescence images to enable label-free cell classification.

High-resolution mitochondria imaging in combination with image analysis tools have significantly advanced our understanding of cellular function in health and disease. However, most image analysis tools for mitochondrial studies have been designed to work with fluorescently labeled images only. Additionally, efforts to integrate features describing mitochondrial networks with machine learning techniques for the differentiation of cell types have been limited. Herein, we present AutoMitoNetwork software for image-based assessment of mitochondrial networks in label-free autofluorescence images using a range of interpretable morphological, intensity, and textural features. To demonstrate its utility, we characterized unstained mitochondrial networks in healthy retinal cells and in retinal cells exposed to two types of treatments: rotenone, which directly inhibited mitochondrial respiration and ATP production, and iodoacetic acid, which had a milder impact on mitochondrial networks via the inhibition of anaerobic glycolysis. For both cases, our multi-dimensional feature analysis combined with a support vector machine classifier distinguished between healthy cells and those treated with rotenone or iodoacetic acid. Subtle changes in morphological features were measured including increased fragmentation in the treated retinal cells, pointing to an association with metabolic mechanisms. AutoMitoNetwork opens new options for image-based machine learning in label-free imaging, diagnostics, and mitochondrial disease drug development.

Double-Clad Antiresonant Hollow-Core Fiber and Its Comparison with Other Fibers for Multiphoton Micro-Endoscopy.

Label-free and multiphoton micro-endoscopy can transform clinical histopathology by providing an in situ tool for diagnostic imaging and surgical treatment in diseases such as cancer. Key to a multiphoton imaging-based micro-endoscopic device is the optical fiber, for distortion-free and efficient delivery of ultra-short laser pulses to the sample and effective signal collection. In this work, we study a new hollow-core (air-filled) double-clad anti-resonant fiber (DC-ARF) as a high-performance candidate for multiphoton micro-endoscopy. We compare the fiber characteristics of the DC-ARF with a single-clad anti-resonant fiber (SC-ARF) and a solid core fiber (SCF). In this work, while the DC-ARF and the SC-ARF enable low-loss (<0.2 dBm-1), close to dispersion-free excitation pulse delivery (<10% pulse width increase at 900 nm per 1 m fiber) without any induced non-linearities, the SCF resulted in spectral broadening and pulse-stretching (>2000% of pulse width increase at 900 nm per 1 m fiber). An ideal optical fiber endoscope needs to be several meters long and should enable both excitation and collection through the fiber. Therefore, we performed multiphoton imaging on endoscopy-compatible 1 m and 3 m lengths of fiber in the back-scattered geometry, wherein the signals were collected either directly (non-descanned detection) or through the fiber (descanned detection). Second harmonic images were collected from barium titanate crystals as well as from biological samples (mouse tail tendon). In non-descanned detection conditions, the ARFs outperformed the SCF by up to 10 times in terms of signal-to-noise ratio of images. Significantly, only the DC-ARF, due to its high numerical aperture (NA) of 0.45 and wide-collection bandwidth (>1 µm), could provide images in the de-scanned detection configuration desirable for endoscopy. Thus, our systematic characterization and comparison of different optical fibers under different image collection configurations, confirms and establishes the utility of DC-ARFs for high-performing label-free multiphoton imaging-based micro-endoscopy.

Multi-Illumination Single-Holographic-Exposure Lensless Fresnel (MISHELF) Microscopy: Principles and Biomedical Applications.

Lensless holographic microscopy (LHM) comes out as a promising label-free technique since it supplies high-quality imaging and adaptive magnification in a lens-free, compact and cost-effective way. Compact sizes and reduced prices of LHMs make them a perfect instrument for point-of-care diagnosis and increase their usability in limited-resource laboratories, remote areas, and poor countries. LHM can provide excellent intensity and phase imaging when the twin image is removed. In that sense, multi-illumination single-holographic-exposure lensless Fresnel (MISHELF) microscopy appears as a single-shot and phase-retrieved imaging technique employing multiple illumination/detection channels and a fast-iterative phase-retrieval algorithm. In this contribution, we review MISHELF microscopy through the description of the principles, the analysis of the performance, the presentation of the microscope prototypes and the inclusion of the main biomedical applications reported so far.

Amyloid Fibrils of Stefin B Show Anisotropic Properties.

Human stefin B, a member of the cystatin family of cysteine protease inhibitors, tends to form amyloid fibrils under relatively mild conditions, which is why it is used as a model protein to study amyloid fibrillation. Here, we show for the first time that bundles of amyloid fibrils, i.e., helically twisted ribbons, formed by human stefin B exhibit birefringence. This physical property is commonly observed in amyloid fibrils when stained with Congo red. However, we show that the fibrils arrange in regular anisotropic arrays and no staining is required. They share this property with anisotropic protein crystals, structured protein arrays such as tubulin and myosin, and other anisotropic elongated materials, such as textile fibres and liquid crystals. In certain macroscopic arrangements of amyloid fibrils, not only birefringence is observed, but also enhanced emission of intrinsic fluorescence, implying a possibility to detect amyloid fibrils with no labels by using optical microscopy. In our case, no enhancement of intrinsic tyrosine fluorescence was observed at 303 nm; instead, an additional fluorescence emission peak appeared at 425 to 430 nm. We believe that both phenomena, birefringence and fluorescence emission in the deep blue, should be further explored with this and other amyloidogenic proteins. This may allow the development of label-free detection methods for amyloid fibrils of different origins.

Insights into S. aureus-Induced Bone Deformation in a Mouse Model of Chronic Osteomyelitis Using Fluorescence and Raman Imaging.

Osteomyelitis is an infection of the bone that is often difficult to treat and causes a significant healthcare burden. Staphylococcus aureus is the most common pathogen causing osteomyelitis. Osteomyelitis mouse models have been established to gain further insights into the pathogenesis and host response. Here, we use an established S. aureus hematogenous osteomyelitis mouse model to investigate morphological tissue changes and bacterial localization in chronic osteomyelitis with a focus on the pelvis. X-ray imaging was performed to follow the disease progression. Six weeks post infection, when osteomyelitis had manifested itself with a macroscopically visible bone deformation in the pelvis, we used two orthogonal methods, namely fluorescence imaging and label-free Raman spectroscopy, to characterise tissue changes on a microscopic scale and to localise bacteria in different tissue regions. Hematoxylin and eosin as well as Gram staining were performed as a reference method. We could detect all signs of a chronically florid tissue infection with osseous and soft tissue changes as well as with different inflammatory infiltrate patterns. Large lesions dominated in the investigated tissue samples. Bacteria were found to form abscesses and were distributed in high numbers in the lesion, where they could occasionally also be detected intracellularly. In addition, bacteria were found in lower numbers in surrounding muscle tissue and even in lower numbers in trabecular bone tissue. The Raman spectroscopic imaging revealed a metabolic state of the bacteria with reduced activity in agreement with small cell variants found in other studies. In conclusion, we present novel optical methods to characterise bone infections, including inflammatory host tissue reactions and bacterial adaptation.

T Cell Antigen Recognition and Discrimination by Electrochemiluminescence Imaging.

Adoptive T lymphocyte (T cell) transfer and tumour-specific peptide vaccines are innovative cancer therapies. An accurate assessment of the specific reactivity of T cell receptors (TCRs) to tumour antigens is required because of the high heterogeneity of tumour cells and the immunosuppressive tumour microenvironment. In this study, we report a label-free electrochemiluminescence (ECL) imaging approach for recognising and discriminating between TCRs and tumour-specific antigens by imaging the immune synapses of T cells. Various T cell stimuli, including agonistic antibodies, auxiliary molecules, and tumour-specific antigens, were modified on the electrode's surface to allow for their interaction with T cells bearing different TCRs. The formation of immune synapses activated by specific stimuli produced a negative (shadow) ECL image, from which T cell antigen recognition and discrimination were evaluated by analysing the spreading area and the recognition intensity of T cells. This approach provides an easy way to assess TCR-antigen specificity and screen both of them for immunotherapies.

Label-Free Imaging of Membrane Potentials by Intramembrane Field Modulation, Assessed by Second Harmonic Generation Microscopy.

Optical interrogation of cellular electrical activity has proven itself essential for understanding cellular function and communication in complex networks. Voltage-sensitive dyes are important tools for assessing excitability but these highly lipophilic sensors may affect cellular function. Label-free techniques offer a major advantage as they eliminate the need for these external probes. In this work, it is shown that endogenous second-harmonic generation (SHG) from live cells is highly sensitive to changes in transmembrane potential (TMP). Simultaneous electrophysiological control of a living human embryonic kidney (HEK293T) cell, through a whole-cell voltage-clamp reveals a linear relation between the SHG intensity and membrane voltage. The results suggest that due to the high ionic strengths and fast optical response of biofluids, membrane hydration is not the main contributor to the observed field sensitivity. A conceptual framework is further provided that indicates that the SHG voltage sensitivity reflects the electric field within the biological asymmetric lipid bilayer owing to a nonzero χeff(2) tensor. Changing the TMP without surface modifications such as electrolyte screening offers high optical sensitivity to membrane voltage (≈40% per 100 mV), indicating the power of SHG for label-free read-out. These results hold promise for the design of a non-invasive label-free read-out tool for electrogenic cells.

Single-shot quantitative phase imaging as an extension of differential interference contrast microscopy.

Various studies have been conducted to obtain quantitative phase information based on differential interference contrast (DIC) microscopy. As one such attempt, we propose in this study a single-shot quantitative phase imaging (QPI) method by combining two developments. First, an add-on optical system to a commercialized DIC microscope was developed to perform quantitative phase gradient imaging (QPGI) with single image acquisition using a polarization camera. Second, an algorithm was formulated to reconstitute QPI from the obtained QPGI by reducing linear artifacts, which arise in simply integrated QPGI images. To demonstrate the applicability of the developed system in cell biology, the system was used to measure various cell lines and compared with fluorescence microscopy images of the same field of view. Consistent with previous studies, nucleoli and lipid droplets can be imaged by the system with greater optical path lengths (OPL). The results also implied that combining fluorescence microscopy and the developed system might be more informative for cell biology research than using these methods individually. Exploiting the single-shot performance of the developed system, time-lapse imaging was also conducted to visualize the dynamics of intracellular granules in monocyte-/macrophage-like cells. Our proposed approach may accelerate the implementation of QPI in standard biomedical laboratories.

Label-Free Electrochemiluminescence Imaging of Single-Cell Adhesions by Using Bipolar Nanoelectrode Array.

A label-free and fast approach for positive electrochemiluminescence (ECL) imaging of single cells by bipolar nanoelectrode array is proposed. The reduction of oxygen at a platinized gold nanoelectrode array in a closed bipolar electrochemical system is coupled with an oxidative ECL process at the anodic side. For elevating the ECL imaging contrast of single cells, a driving voltage of -2.0 V is applied to in situ generate oxygen confined beneath cells that is subsequently used for ECL imaging at 1.1 V. High oxygen concentration in the confined space resulting from steric hindrance generates prominent oxygen reduction current at the cathodic side and higher ECL intensity at the anodic side, allowing positive ECL imaging of the cells adhesion region with excellent contrast. Cell morphology and adhesion strength can be successfully imaged with high image acquisition rate. This approach opens a new avenue for label-free imaging of single cells.

Time-course quantitative mapping of caffeine within the epidermis, using high-contrast pump-probe stimulated Raman scattering microscopy.

BACKGROUND: An assessment of the drug penetration and distribution profiles within the skin is essential in dermatology and cosmetology. Recent advances in label-free imaging technologies have facilitated the direct detection of unlabeled compounds in tissues, with high resolution. However, it remains challenging to provide quantitative time-course distribution maps of drugs within the complex skin tissue. The present study aims at acquiring the real-time quantitative skin penetration profiles of topically applied caffeine, by means of a combination of pump-probe phase-modulated stimulated Raman scattering (PM-SRS) and confocal reflection microscopy. The recently developed PM-SRS microscopy is a unique imaging tool that can minimize strong background signals through a pulse-shaping technique, while providing high-contrast images of small molecules in tissues. MATERIALS AND METHODS: Reconstructed human skin epidermis models were used in order to analyze caffeine penetration in tissues. The penetration profiles of caffeine in an aqueous solution, an oil-in-water gel, and a water-in-oil gel were examined by combining PM-SRS and confocal reflection microscopy. RESULTS: The characteristic Raman signal of caffeine was directly detected in the skin model using PM-SRS. Integrating PM-SRS and confocal reflection microscopy allowed real-time concentration maps of caffeine to be obtained from formulation samples, within the skin model. Compared with the conventional Raman detection method, PM-SRS lowered the background tissue-oriented signals and supplied high-contrast images of caffeine. CONCLUSION: We successfully established real-time skin penetration profiles of caffeine from different formulations. PM-SRS microscopy proved to be a powerful, non-invasive, and real-time depth-profile imaging technique for use in quantitative studies of topically applied drugs.